The biochemical basis for the addictive properties of opiates may relate to deficiencies in endogenous levels or in the turnover of opioid peptides. It is therefore important to gain as much information as possible on the enzymatic mechanisms responsible for opioid peptide biosynthesis. The synthesis of biologically active opioid peptides requires a series of enzymatic steps beginning with proteolytic cleavage of the precursor and ending with various terminal modifications such as carboxypeptidase B- like trimming and amidation. Probably the most important- and least well understood- step in the biosynthesis of opioid peptides is the proteolytic cleavage of the precursor proteins at basic residue "processing signals". The enzyme(s) which accomplish these trypsin-like proteolytic conversions have not yet been identified with certainty. One of the most promising candidate enzymes is a serine protease which is colocalized with enkephalins within adrenal chromaffin granules. The proposed experiments are designed to provide a better understanding of the role of this enzyme in opioid peptide biosynthesis. We plan to purify this enzyme from adrenal and pituitary tissue, develop new substrates and inhibitors for the study of this and potentially other opioid peptide processing enzymes, and take advantage of recombinant DNA techniques to compare the reactivity of this enzyme with all three opioid peptide precursors. The knowledge gained from these studies will advance our understanding of the enzymatic mechanisms involved in opioid peptide formation and may ultimately aid us in the rational design of enzyme inhibitors in the therapeutic treatment of opiate addiction.